The objectives of this research plan are distinctly twofold: the first is to purify the enzyme, insulin specific protease of rat liver to homogeneity and then to determine the points of proteolytic cleavage within the insulin molecule. The second objective is to purify to homogeneity the major protein inhibitor of the enzyme present in human plasma, to determine its mode of action and then its amino acid sequence. These objectives are aimed at understanding and validating the mechanism of action of the enzyme. The methods to be used in the enzyme purification are the conventional techniques of calcium phosphate gel adsorption and elution, gel filtration on Sephadex G-100 and ion-exchange chromatography upon QAE Sephadex and hydroxyl apatite, but carried out very rapidly with enzyme storage in liquid nitrogen in between these procedures. If necessary to achieve homogeneity affinity chromatography will finally be carried out upon Sephadex G-200 linked to insulin through a ligand. The pure insulin specific protease enzyme will be incubated with insulin and the peptides released will be extracted with trichloroacetic acid (TCA). After removal of TCA and reaction with 5-dimethylaminonaphthalene sulfonyl(dansyl) chloride, the peptides will be separated by polyamide thin-layer chromatography, each peptide hydrolyzed, the N terminal amino acid and qualitative amino acid composition determined by the dansyl chloride method. This should allow unequivocal assignment of the points of attack within the hormone. The methods which have proven of value in the purification and characterization of the human alpha1 globulin insulin-specific-protease inhibitors from commercially available material are the following: heat denaturation and precipitation of noninhibitory protein, gel filtration upon Sephadex G-100 and ion-exchange chromatography upon QAE Sephadex. The alpha1 globulin preparation obtained from the National Fractionation Center of the ARC Blood Research Laboratories is in a more native state requiring different techniques such as alcohol and/or acid-alcohol extraction prior to the use of the above-mentioned techniques used with the commercial preparation.